A normal b-globin allele as a modifier gene ameliorating the severity of a-thalassemia in mice

Thalassemia is a heritable human anemia caused by a variety of mutations that affect expression of the aor the b-chain of hemoglobin. The expressivity of the phenotype is likely to be inf luenced by unlinked modifying genes. Indeed, by using a mouse model of a-thalassemia, we find that its phenotype is strongly inf luenced by the genetic background in which the a-thalassemia mutation resides [129svyevy129svyev (severe) or 129svyevyC57BLy6 (mild)]. Linkage mapping indicates that the modifying gene is very tightly linked to the b-globin locus (Lod score 5 13.3). Furthermore, the severity of the phenotype correlates with the size of b-chain-containing inclusion bodies that accumulate in red blood cells and likely accelerate their destruction. The b-major globin chains encoded by the two strains differ by three amino acids, one of which is a glycine-to-cysteine substitution at position 13. The Cys-13 should be available for interchain disulfide bridging and consequent aggregation between excess b-chains. This normal polymorphic variation between murine b-globin chains could account for the modifying action of the unlinked b-globin locus. Here, the variation in severity of the phenotype would not depend on a change in the ratio between aand b-chains but on the chemical nature of the normal b-chain, which is in excess. This work also indicates that modifying genes can be normal variants that—absent an apparent physiologic rationale—may be difficult to identify on the basis of structure alone. The severity of thalassemia, one of the most prevalent of the heritable human anemias, is brought about by the insufficient production of functional hemoglobin and the degree of quantitative imbalance between aor b-globin chains (1–8). Needless to say, there are a large number of genetic alterations that can cause such a disturbance in globin chain production, and many of these have been characterized at the molecular level (see review in ref. 1). Thus, for example, thalassemias arise through mutations that induce alterations in gene transcription, pre-mRNA splicing, mRNA and protein stability, polyadenylation, and even translation (1). The disease offers a virtual textbook of potential molecular genetic lesions. In addition to the great variety of mutations that affect the expressivity of the phenotype, the phenotype is likely to be further influenced by unlinked modifying genes that could account for the variable severity of the disease seen in certain families wherein the primary genetic lesion is likely to be identical by descent (9–11). To date, save for pathologic hemaglobinopathies, no such modifying genes have been identified. An opportunity to address the issue of modifying genes arose when we created a mouse model of a-thalassemia and noted that the severity of the disease was greatly influenced by the genetic background in which the mutation was expressed [129svyevy129svyev (severe) or 129svyevyC57BLy6 (mild)] (12). In the work described below, we have identified the modifying gene that ameliorates the a-thalassemia phenotype as the normal b-globin allele present in the C57BLy6 strain of mouse. The severe form of thalassemia can be related to a Cys-13 residue present in the b-globin gene of the 129svyev strain and the milder form to a glycine in this position in the C57BLy6 strain. The aggregates of excess b-chains that likely form via cysteine sulfhydryl bridges would account for the very large inclusion bodies seen in the red blood cells in a-thalassemic mice bearing two copies of the 129svyev b-globin allele. This mechanism indicates that the severity of thalassemia can depend not only on the imbalance between aand b-chains but on the nature of the allelic form of the chain in excess. MATERIALS AND METHODS Animals. The a1 knockout mice on 129svyev background used for the genetic crosses were generated in our laboratory (12). The inbred strains C57BL-6 and BALByc were obtained from Taconic Farms and were housed in our animal facility until they were old enough to breed. Genotyping. Southern blot analysis. Tail DNAs were prepared from tail biopsies as described (12). DNAs were then digested with EcoRI restriction endoclease run on 1% agarose gel, transferred to nitrocellulose membrane, and hybridized with b-globin cDNA derived from a mouse spleen cDNA library. Analysis of the a-globin genotype was as described (12). PCR. D7Mit-40 primers were used in a PCR reaction to distinguish between b-globins of C57BLy6, 129svyev, and BALByc alleles. The amplification reaction contained 1 ml of diluted DNA (1:10 volyvol in H20), 17 ml of PCR supermix (GIBCOyBRL), 1.5 ml of 59 oligonucleotides and 1.5 ml of 39 oligonucleotides in a 21-ml total reaction volume. The thermal cycle conditions were: 94°C for 1 min followed by 35 cycles of 94°C for 30 sec, 52°C for 30 sec, and extension at 72°C for 1 min. The PCR reactions were analyzed by gel electrophoresis in a 4% agarose (NuSieve GTG) gel, prepared, and run with 13 TBE (0.1 M Trisy0.08 M boratey0.001 M EDTA, pH 8.6). Peripheral Blood Analysis. Blood was collected from tails in potassium EDTA-treated microtubules. Hematologic indices were determined by using standard hospital methods in the clinical laboratory at Children’s Hospital, Boston. Cloning Adult b-Globins by PCR. Tail DNA from 129svyev mice was used as a template for a PCR reaction. Primers were designed according to a published BALByc b-globin major (13). An EcoRI site was added to the primers (forward) 59-ACGAATTCATGGTGCACCTGACTGAT-39 and (reverse) 59-ACGAATTCGTGGTACTTGTGAGCCAAGCA-39. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. †To whom reprint requests should be addressed. e-mail: leder@ rascal.med.harvard.edu.